Chondrocyte phenotype-specific differences in cellular calcium metabolism and its regulation by autocrine calcitropic systems have been identified and characterized in the prior funding period. PTHrP has recently been demonstrated to be a critical autocrine regulator of chondrocyte maturation. PTHrP functions as both a mitogen and a suppressor of hypertrophy, allowing controlled chondrocyte proliferation essential to longitudinal bone growth. However, the exact mechanism of how chondrocytes hypertrophy, and how this is regulated has been, up to now, a serious gap in our knowledge. PTHrP elevates cytosolic calcium in chondrocytes and, in turn, appears to be negatively regulated by cytosolic calcium. Therefore, PTHrP may initiate the progressive rise in calcium which subsequently shuts off its expression in an appropriate temporal sequence to stop proliferation and allow hypertrophy. PTHrP also inhibits Na+ transporters which drive cell volume increase. The research proposed in Specific Aim 1 will investigate the relationship of PTHrP and chondrocyte cytosolic calcium. The applicants will: (A) identify regulation of PTHrP transcripts and protein by manipulation of cytosolic calcium; (B) determine if the PTHrP promoter contains regulatory regions which are calcium-sensitive; and (C) investigate the regulation of annexin V by PTHrP to determine if this is the mechanism by which PTHrP increases calcium levels. Specific Aim 2 will investigate the novel finding of ion transport mechanisms which regulate cell volume in cartilage, in order to determine: (A) which mechanisms are present, using acute volume regulation and molecular screening approaches; and (B) ion transport activities and levels as a function of chondrocyte maturation. The investigators will focus on two Na+-dependent transporters strongly implicated by their preliminary data as mechanistically involved in hypertrophy: NHE1 (Na+/H+ exchanger 1), and NKCC1 (Na+-K+-2C1-cotransporter 1). In Specific Aim 3, plans are advanced to: (A) investigate the regulation of NHE1 and NKCC1 by PTHrP; (B) determine the effect of inhibiting these mechanisms on cell swelling and expression of hypertrophic phenotype; and (C) use molecular approaches of transient transfections and antisense strategies to confirm the functional importance of NHE1 and NKCC1 in chondrocyte hypertrophy. Preliminary data has allowed the applicant to formulate a new theory of how chondrocyte hypertrophy occurs at a molecular level, and the proposed research will test the component mechanisms of this model. The principal investigator believes that significant and fundamental new information on chondrocyte biology will result from the project.